Project Summary Interleukin-1 Receptor (IL-1R)-Associated Kinase 1, IRAK1, is a core transducer of Toll-like receptor (TLR) and IL-1R-mediated innate immune signaling from flies to humans. In response to pathogen infection, ligated TLR/IL-R receptors almost instantaneously activate IRAK1 via formation of the MyDDosome complex (IL-1R/TLR?MyD88-IRAK4-IRAK1) at the cell surface. Once phosphorylated by IRAK4 and itself, fully activated IRAK1 dissociates from the platform and engages NF-kB and other signaling cascades, culminating in the acute inflammatory response. Until recently, vertebrate IRAK1 had not been implicated in processes other than the microbial response. In an unbiased zebrafish screen, we recently identified IRAK1 as essential for cell survival in response to ionizing radiation (gIR) (Liu et al., Nat Cell Biol 2019; ref. 1). This function is conserved in human cells and drives cellular resistance to radiotherapy (R-RT) in tumor models. Rather than acting to stimulate NF-kB, IRAK1 drives cell survival by countering apoptosis mediated by the PIDDosome complex (PIDD-RAIDD- caspase-2). In further contrast with canonical IRAK1/4 immune signaling, our preliminary data indicate that the IRAK1 response to gIR: (i) fully requires its kinase activity; (ii) does not require the IL-1R/TLR?IRAK1/4 adaptor MyD88; and most strikingly, (iii) initiates in the nucleus of irradiated cells and not at the cell surface. gIR-induced IRAK1 activation does however occur within minutes of stimulus and absolutely requires IRAK4, suggesting the existence of a novel oligomeric platform responsible for orchestrating gIR-induced IRAK1 activation in place of the MyDDosome. While the evidence convincingly points to a novel IRAK1 stress response pathway in vertebrates, the cellular and molecular bases of gIR-induced IRAK1 activation remain to be defined. In Aim 1, we will monitor the localization of both active and native IRAK1 as a function of time after gIR; identify the cellular signal(s) that effectively trigger IRAK1 activation in irradiated cells, with DNA breaks, micronucleation, cytokines and danger- associated molecular patterns (DAMPs) as primary candidates; and explore whether environmental stresses (e.g., UV irradiation) can trigger the pathway. In Aim 2, we will dissect the molecular mechanism of IRAK1 activation in response to gIR, first by focusing on the roles of IRAK4 and IRAK1 itself; second by identifying the DD protein substituting for MyD88 as scaffold for the gIR-induced IRAK4/IRAK1 activation platform; and finally taking unbiased, larger scale proteomic approaches toward the unbiased identification of the upstream sensors, transducers and regulators that orchestrate IRAK1 activation in irradiated nuclei. Beyond illuminating a novel stress response pathway in vertebrates, our proposal explores a pathway implicated in tumor R-RT. Thus, an additional immediate impact of our work might be the discovery of novel drug targets for overcoming R-RT in the majority of cancer patients that receive RT as part of their treatment.